Method of detecting disease conditions in C. elegans infected with animal virus

ABSTRACT

The present invention provides methods for studying pathogenesis of mammalian viruses. In particular, the present invention provides a nonhuman animal model system for studying disease mechanism wherein the nonhuman animal model is infected with an animal virus. In a preferred embodiment, the animal model is  C. elegans  and the animal virus is vesicular stomatitis virus (VSV).

1. RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/435,806, filed on Dec. 22, 2002.

2. FIELD OF INVENTION

The present invention relates to animal models and methods for studyingpathogenesis of mammalian viruses. More specifically, the presentinvention provides a disease model of Vesicular Stomatitis Virusinfection with C. elegans and methods for studying disease phenomenon.

3. BACKGROUND OF THE INVENTION

Effective design of antiviral therapeutics, diagnostic devices andvaccines requires understanding viral mechanism of disease pathogenesis.Presently, viral replication and disease mechanisms are studied eitherin cell culture or vertebrate hosts such as rodents (mice, rats, guineapigs) or nonhuman primates. However, cell culture systems do not provideproper models for studying disease mechanisms involving physiologicalresponses of multicellular origins such as the inflammatory process orinnate immune responses or viral induced immunopathologies. Viralpathogenic studies in intact vertebrate hosts such as mice presentdifficulties due to many factors including the number of animalsrequired for statistically significant results, long experimentalduration times and high expense.

Disease development upon viral infection is highly dependent on thegenetics and physiology of the host. Viruses are obligate intracellularparasites and depend on various components of the host machinery fornearly all aspects of their life cycle, including replication, geneexpression and cell-to-cell spread. As viruses manipulate the hostcellular and systemic physiology, host responses lead either to diseasedevelopment or recovery. For mammalian animal viruses, previous studiesof host factors conferring susceptibility or resistance to viralinfection have been limited to in vitro host models using infected cellsor tissue culture or in vivo models involving experimental infection ofprimate or small animal mammalian hosts. Both in vitro and in vivomodels of viral infection have major disadvantages. Mammalian models,namely mouse or primates, are costly and technically demanding. Samplesizes are often limited. In addition, genetic manipulation of the hostgenome is technically difficult and often requires long generationtimes. In vitro tissues and/or cell culture addresses many of theseproblems but the ability to conduct studies on disease aspects thatdepend on the physiological responses of multiple organs or cellularsystems. Therefore, a simple animal model system capable of elucidatingviral disease mechanisms of mammalian systems is desired.

The nematode Caenorhabditis elegans (C. elegans) is an attractive modelsystem for studying viral disease for a number of technical andscientific reasons. In particular, the complete genome has beensequenced and a well characterized genetic map and a collection ofmutants are available. Moreover, the C. elegans' fast generation timeand simple and inexpensive maintenance makes it relatively easy toobtain large sample sizes for experimental studies. Previous studieswith C. elegans as models for bacterial pathogenesis demonstrated adisease phenotype in the nematode upon exposure to certain strains ofPsuedomonas, a facultative intracellular bacterial pathogen.(Mahajan-Miklos et al., 199 Cell 96:47-56 and Tan et al., 1999 Proc.Natl. Acad Sci. USA 96:2408-2413). Disease occurrence was dependent onseveral bacterial genes previously shown to be necessary for virulencein other infection models. In addition, several new virulence-associatedgenes were identified. These initial studies were extended to show thatother facultative intracellular bacterial pathogens, includingSalmonella (Labrousse et al., 2000 Current Biology 10: 1543-1545) and avariety of gram-positive pathogens (Garsin et al., 2000 Proc. Natl. AcadSci. USA 98:10872-10877) can also induce disease in C. elegans. Otherstudies have focused on host defense mechanisms involved in controllingbacterial infections (Pujol et al., 2001 Current Biology 11:809-821).Random mutagenesis screens have identified components of themitogen-activated protein kinases (MAPK) signaling pathway as beinginfluential in host susceptibility to bacterial infection in thenematode (Kim et al., 2002 Science 297:623-626). Yet, no studies havedemonstrated disease susceptibility of C. elegans by animal viruses notknown to infect C. elegans until the present invention. Accordingly, thepresent invention provides a simple model system that can be used tostudy disease pathogenesis of mammalian viruses.

Discussion or citation of a reference herein shall not be construed asan admission that such reference is prior art to the present invention.

4. SUMMARY OF THE INVENTION

The present invention relates to the inventors' unexpected ability todemonstrate that vesicular stomatitis virus (VSV) can cause disease inC. elegans, an invertebrate which is not known to be infected by VSV orother vertebrate animal viruses. VSV is an enveloped virus with anegative-sense RNA genome that must be transcribed by a virus-encodedRNA-dependent RNA polymerase to replicate the viral RNA genome and toproduce mRNAs which are used in synthesis of viral proteins. VSVlethally infects vertebrate animal cells in in vitro culture and viralinfection of whole animal such as mice causes disease.

Therefore, the present invention provides an animal model for studyingdisease mechanisms comprising C. elegans infected with an animal virus.In a preferred embodiment, C. elegans is infected with VSV.

The C. elegans can be wild-type, mutant or transgenic. Moreover, thetransgenic C. elegans may comprise a reporter gene such as but notlimited to luciferase or the green fluorescent protein wherein theexpression of the reporter protein is dependent on the presence of viralgene expression or viral infection.

Potential therapeutics that target symptom pathways may interfere withthe development of diseases but do not significantly effect viralreplication. Therefore, the present invention provides an infectionmodel system for screening agents that interact with diseasedevelopment. In one embodiment of the present method, C. elegansinfected with an animal virus is combined with an agent and the effectof the agent on a phenomenon associated with viral disease mechanisms isdetermined.

In another embodiment, the present invention provides a method foridentifying host genes that are involved in disease development.

In still another embodiment, the present invention provides a method forstudying disease conditions comprising infecting C. elegans with VSV anddetecting an abnormal phenotype in the infected C. elegans which is notobserved in healthy uninfected C. elegans. These phenotypes which mayvary with the disease conditions, include but are not limited todecreased numbers of progeny, altered larval development, muscleabnormalities leading to uncoordinated movement or egg-laying defects,altered lifespan of the adult C. elegans and death.

5. DESCRIPTION OF THE FIGURES

FIG. 1 demonstrates that vesicular stomatitis virus (VSV) infectiondecreases C. elegans population size and skews larval development.Individual L3 larvae were cultured in liquid media in the presence orabsence of 10⁶ PFU/ml VSV fluorescent protein (VSV-GFP) recombinantvirus. After 3 days at 25° C., total progeny and number perdevelopmental stage were counted.

FIG. 2 demonstrates that infectious VSV is responsible for the decreasein population size in C. elegans. Individual L3 larvae were cultured inliquid media in the absence of virus, in the presence of 10⁶ PFU/ml VSVfluorescent protein (VSV-GFP) recombinant virus or in the presence ofthe equivalent amount of UV-inactivated VSV-GFP. After 3 days at 25° C.,total progeny and number per developmental stage were counted.

FIG. 3 demonstrates fluorescent and phase-contrast images of cellsisolated from C. elegans embryos that were infected with vesicularstomatitis virus-green fluorescent protein (VSV-GFP) recombinant virus.Cells were incubated with the recombinant virus for 12 hours at 25° C.at a multiplicity of 10 infectious unit per cell. At 48 hourpost-infection, the cells were examined by fluorescent microscopy forGFP expression.

FIG. 4 demonstrates that VSV does not infect normal muscle cells. Cellswere isolated from embryos of a transgenic C. elegans strain whichexpresses GFP in C. elegans muscle cells. Cells were infected with VSVrecombinant virus which expresses the dsRED variant of GFP. The greenfluorescent cells represent muscle cells and the red fluorescent cellsrepresent VSV infected cells.

FIG. 5 demonstrates that VSV infected C. elegans can synthesize mRNAsthat encode the viral N protein sequence. RNA was isolated from virusinfected C. elegans and baby hamster kidney (BHK) cells. Infected anduninfected BHK cells were used as positive and negative assay controls,respectively. cDNA synthesis was made using oligonucleotide primers thatspecifically hybridized with the N protein mRNA transcripts and not tothe viral RNA genome. The resultant cDNA sequences were amplified by PCRusing oligonucleotide primers specific for N protein gene sequences. PCRsignals were observed only in the infected C. elegans and BHK cells.

FIG. 6( a) demonstrates that cells isolated from a mutant C. elegansstrain rde-1 that contains a defective gene in the RNA interference(RNAI) pathway can be infected with VSV.

FIG. 6( b) demonstrates that the mutant C. elegans strain rde-1 is moresusceptible to VSV infection than an unmutated wild type C. elegansstrain.

6. DETAILED DESCRIPTION OF THE INVENTION

This section presents a detailed description of the invention and itsapplications. This description is by way of several exemplaryillustrations, in increasing detail and specificity, of the generalmethods of this invention. These examples are non-limiting and relatedvariants will be apparent to one of skill in the art.

The present inventors demonstrated that disease phenomenon can begenerated in C. elegans upon exposure to an animal virus. Therefore, thepresent invention provides an animal model for studying diseasemechanisms comprising C. elegans infected with an animal virus. C.elegans of this invention may be wild-type or variants. In particular,variants of C. elegans include transgenic or mutant C. elegans. In oneembodiment, the C. elegans of the present animal model is the wild-typeC. elegans. In another embodiment, the C. elegans of the present animalmodel is a transgenic C. elegans. Transgenic C. elegans have foreigngenes incorporated into the C. elegans genome. The foreign genes mayinclude but are not limited to human genes, mammalian genes or reportergenes. Transgenic C. elegans which are expressing reporter genes areconstructed such that the reporter gene is expressed only under certainconditions. Mutant C. elegans have mutant C. elegans genes. The term“transgene” is used herein to describe genetic material that has been oris about to be artificially inserted into the host cell, particularly acell of a living animal. The transgene is used to alter cells such thata permanent or transient genetic change is induced in a cell followingincorporation of exogenous DNA. A permanent genetic change is generallyachieved by introduction of he foreign DNA into the genome of the cell.

Studies using the present invention revealed that C. elegans rde-1mutants defective in the RNA interference (RNAi) pathway are moresusceptible to VSV infection than the wild-type C. elegans (see FIGS. 6a/b). Therefore, in another embodiment of the present invention, the C.elegans of the present animal model is a mutant C. elegans.

The present invention also provides an in vitro infection systemcomprising of C. elegans cells which have been isolated from C. eleganshosts and grown in culture which are infected with an animal virus.

Until the present invention, no reports have shown that a mammalianvirus can infect C. elegans. Any potential virus would have to be ableto replicate at temperatures used for nematode culture (25° C.), utilizecellular receptors and machinery sufficiently conserved ininvertebrates, and have an infection cycle acute enough to cause diseasein the short generation time of the host. The obligate intracellularlifestyles of viruses make them highly dependent on the host cell.Because of this, many viruses have very specific cellular tropisms andnarrow host ranges. The present invention provides an animal model forstudying disease mechanism comprising C. elegans infected with obligateintracellular bacteria pathogens. In one embodiment, the bacterialpathogens are selected from a group consisting of Chlamydia, rickettsiaand ehrlichia. The present invention further provides an animal modelfor studying disease infection comprising C. elegans infected withobligate intracellular parasites. In particular, the parasites areselected from a group consisting of toxoplasma and malaria. Moreover,the obligate intracellular bacteria require the eukaryotic host to growwhereas the facultative bacteria like the pseudomonas can be propagatedby other growth conditions without the eukaryotic cells. Therefore, thepresent invention also provides an animal model for studying diseaseinfection comprising C. elegans infected with obligate intracellularpathogens including fungi and mold.

The present invention demonstrated that C. elegans infected withvesicular stomatitis virus (VSV) revealed decrease in progeny and skeweddevelopment (see FIG. 1). Therefore, the present invention provides ananimal model for studying disease mechanism comprising C. elegans thatare infected with VSV or its variants. Vesicular stomatitis virusvariants include recombinant VSV or mutant VSV. A recombinant VSV maycontain a reporter gene. Various reporter genes will be apparent to oneskilled in the art.

The vesicular stomatitis virus envelope protein known as VSV G proteinis the protein on the VSV virion which binds to a receptor on the cellsurface to initiate infection. The viral envelope protein participatesin virus binding to and/or entry of the infectious virus into a targetcell. The term “viral envelop protein” refers to the protein(s) embeddedin the membrane which surrounds the viral nucleocapsid. Viruses mustencode viral proteins which recognize receptors on the host cell toinitiate infection. Therefore, the envelop proteins of other unrelatedviruses can be incorporated into the membrane of VSV particles toreplace the VSV G proteins, thereby requiring infection to be initiatedby the envelop protein recognizing specific receptor on the cellsurface.

In one embodiment, the present invention provides an animal for studyingdisease mechanism comprising C. elegans infected with VSV wherein the Gprotein of the VSV is replaced with the envelop protein of anothervirus. The G protein of VSV can be replaced with the “envelope” proteinsof other viruses such as Ebola, Hepatitis C or HIV. In anotherembodiment, the animal model comprises C. elegans infected with VSVwherein the VSV G protein has been incorporated into the virion membraneof a virus other than VSV.

The present invention provides a method for studying disease mechanismcomprising C. elegans infected with chimeric recombinant virusescharacterized by expression of virus particles containing VSV G proteinin the viral membrane and viral genome containing non-VSV genomicsequence within the particle. The VSV G protein can be transientlyincorporated into the virion membrane of other unrelated viruses by astrategy known as “pseudotyping”. This strategy is useful for studyingthe replication of viruses for which cell lines are not available.Successful replication of the non-VSV viral genomes in C. elegans afterintroduction via VSV pseudotyping again provides a strategy forscreening anti-virals or therapeutic targets when standard cell culturesystems are not available. The VSV G can be continually incorporatedinto the virion membrane for each round of cells infected byconstructing a recombinant virus genome containing the VSV G proteinenvelop gene sequence. The chimeric viruses generated by eitherpseudotyping or recombinant methods can be used to study differentaspects of the viral replication cycle for virus entry into cells or foreffecting disease development in the animal. Knowledge of host genesthat effect viral infection or disease development is important toidentify potential physiological responses and/or pathways that areaffected upon viral infection. These host genes and physiologicalpathways are also important targets for antiviral agents and vaccines.

In another embodiment, the animal model system comprises mutant C.elegans infected with chimeric virus pseudotypes characterized byexpression of virus particles containing VSV G protein in the viralmembrane and viral genome containing non-VSV genome sequences within theparticle.

The present invention provides method for studying disease conditionscomprising infecting C. elegans with an animal virus and detecting aphenotype in the infected C. elegans. The phenotype of diseaseconditions include but are not limited to altered numbers of progeny orbrood sizes, alteration in larval development or adult life span, muscleabnormalities leading to uncoordinated movements or egg-laying defects,altered responses to environmental stimuli such as temperature andodorants. The presence of any of these phenotypes or combinations ofphenotypes is indicative of disease conditions. One skilled in the artcan implore many biological and biochemical techniques such asimmunochemistry, differential interference contrast and fluorescencemicroscopy to examine the physiological changes of the C. elegans.

The present invention also provides a method for studying diseaseconditions comprising infecting C. elegans with VSV; and detecting achange in phenotype of the infected C. elegans wherein change inphenotype is indicative of disease conditions. VSV may be wild-type,mutant or transgenic variants.

VSV G infected C. elegans infection grown in liquid in media at 25° C.for 24 hours displayed disease phenotypes including decreased numbers ofprogeny, defects in larval development, muscle abnormalities leading touncoordinated movement or egg-laying defects and death. Therefore, inone embodiment, the change in phenotype includes decreased numbers ofprogeny, alteration in larval development or adult life-span, muscleabnormalities leading to uncoordinated movement or egg-laying defectsand death.

The present invention provides a method for identifying host genes thatare important for viral infection. In one embodiment, transgenic C.elegans containing a mammalian gene is used to identify a foreign hostprotein that is important for virus replication. In particular, thepresent invention provides a method identifying genes important fordevelopment of disease conditions comprising infecting a C. elegans withan animal virus; and detecting a change in phenotype of the infected C.elegans wherein an increase in a phenomenon associated with viraldisease mechanism is indicative of disease conditions; and identifyinggene that are either induced or repressed by infection of the C.elegans. Once these genes have been identified then their mammaliancounterparts can be tested to see whether the mammalian host genes areimportant for viral replication or disease production.

The present invention can be used to study aspects of innate immunitythat affect the ability of the host to combat viral infection. Standardmutagensis protocols can be used to isolate and characterize mutantstrains of C. elegans that are more susceptible or resistant to VSVinfection. Random mutagenesis will allow for large-scale screens of theC. elegans genome to select for those host mutants that appear todevelop more or less sever disease phenotypes. Many of the C. elegansmutations may be in the host factors or pathways utilized by the virusduring the infectious cycle. Gene mutations that contribute to the hostdefense mechanisms including the innate immune system may be identifiedfrom this screen. The mutant screen may identify new genes or newfunctions for previously known genes.

Replacement of the VSV G protein with other viral envelop proteins(hepatitis C, Ebola, West Nile Virus) means that infection is dependenton the ability of the new envelop protein to bind a receptor t initiateinfection. Once the viral genome is in the cell because it is the VSVgenome that presence of VSV replication is a signal than the relevantenvelop protein recognized its receptor on the C. elegans. Replacementof the VSV genome with other viral genomes allows identification of hostgenes which are important for the replication of the viral genome ofinterest. Therefore, the present invention provides a method foridentifying genes important for development of disease conditionswherein the animal virus is VSV. In particular, the present inventionprovides a method identifying genes important for development of diseaseconditions comprising infecting a C. elegans with vesicular stomatitisvirus; and detecting a change in phenotype of the infected C. eleganswherein an increase in a phenomenon associated with viral diseasemechanism is indicative of disease conditions; and identifying gene thatare either induced or repressed by infection of the C. elegans. Thephenomenon associated with viral disease mechanism includes decreasednumbers of progeny, defects in larval development, alteration in adultlife-span, muscle abnormalities leading to uncoordinated movement oregg-laying defects and death.

In many instances, the limiting step for studying viral pathogenesis isthe ability to identify host genes that are important for the diseaseprocess. A major barrier to host infection by a virus is the requirementfor the expression of specific receptors on the host cell surface whichare recognized by a virus to initiate entry into the cell. Therefore,identification of the host gene(s) which encode the receptor for a virusis an important determinant of the infection process. The presentinvention provides a method for identifying genes important fordevelopment of disease conditions by infecting a mutant C. elegans withan animal virus; detecting a change in the phenotype of the infected C.elegans wherein an increase in a phenomenon associated with viraldisease mechanism is indicative of disease conditions; and identifyinggene that are either induced or repressed by infection of the mutant C.elegans.

Recombinant or chimeric viruses can be used to screen for antiviralssuch as but not limited to inhibitors of the fusion function of theenvelop protein or receptor blocker's which could prevent infection.Other non-VSV viral proteins can be expressed in the background of heVSV recombinant viruses to identify the cognate interaction host factorsas well as evaluate their potential effects on disease development. Inone embodiment, the non-VSV viral proteins are apoptosis mediators. Inyet, another embodiment, the non-VSV viral proteins are proteaseinhibitors.

The C. elegans host may comprise alterations to endogenous genes. Thehost animal may be “knockouts” for a target gene comprising a partial orcomplete loss of function in one or all alleles of an endogenous gene ofinterest. In a knockout, the target expression is undetectable orinsignificant. For example, a knockout of a receptor protein gene meansthat expression of the receptor protein is not detectable and functionof the receptor protein is ablated.

Another limiting step for studying viral replication is the availabilityof suitable cell lines or animals. Suitable cell lines or animals can beobtained by generating transgenic C. elegans strains which expressforeign proteins necessary for virus infection. This may be incombination with a knockout of the C. elegans endogenous gene, whileintroducing an exogenous foreign gene. The present invention furtherprovides a strategy for generating an animal host model for viruseswhich cannot be easily grown or studied in cell culture by constructingmulti-transgenic C. elegans which express a combination of importanthost determinants (receptor and intracellular host factors). Forexample, if a virus does not infect C. elegans, because the nematodedoes not express an appropriate receptor, genes from a susceptible hostsuch as a mouse could be screened in C. elegans to identify receptors.

Potential therapeutics that target disease symptom pathways mayinterfere with disease development while not significantly effectingviral replication. Therefore, another embodiment of the presentinvention provides a method for screening agents that effect viraldiseases comprising combining an agent with transgenic C. eleganswherein the transgenic C. elegans are characterized by expression of atransgenic nucleotide sequence encoding a VSV G protein and determiningthe effect of the agent in the transgenic C. elegans on a phenomenonassociated with viral disease mechanism. The present invention providestherapeutic agents produced using the present method of screening foragents for effect on viral diseases comprising infecting C. elegans withan animal virus; combining an agent with the infected C. elegans; anddetermining the effect of the agent on a phenomenon associated.

In another embodiment, the present method of screening for agents foreffect on viral diseases comprising infecting C. elegans with VSV;combining an agent with the infected C. elegans; and determining theeffect of the agent on a phenomenon associated with viral diseasemechanisms wherein the expression of the reporter protein is decreased.

In yet another embodiment, the present invention provides a method fortesting an agent for effectiveness against a disease conditioncomprising obtaining a C. elegans infected with an animal virus whereinthe C. elegans exhibits a disease condition phenotype; delivering theagent to the C. elegans; and analyzing the effectiveness of the agent onthe C. elegans wherein an agent that diminishes the phenotype isindicative of an agent that has effectiveness against the diseasecondition. The phenotype is selected from the group consisting ofdecreased of number of progeny, defects in larval development,alteration in adult life-span, muscle abnormalities leading touncoordinated movement or egg-laying defects and death. The presentinvention also provides therapeutic agents produced using the presentmethod of testing an agent for effectiveness against a diseasecondition.

The G protein of VSV can be replaced with the “envelope” proteins ofother viruses such as Ebola or HIV. This provides a method foridentifying the host receptor for viruses which either do not grow wellor present unusual growth or biohazard containment issues. Theserecombinant viruses can be used to screen for antivirals such asinhibitors of the function of the envelope protein or “receptor”blockers” which could prevent infection. Other non-VSV viral proteinscan be expressed in the background of the VSV recombinant viruses toidentify the cognate interacting host factors as well as evaluate theirpotential effects on disease development. In one embodiment, non-VSVviral proteins are selected from the group consisting of serpin andapoptosis mediators.

The present invention can also be used to study the infection mechanismand defense mechanisms of mutant strains. These mutant strains containmutations in genes including the defensin-like antimicrobial peptides,Toll-signaling receptors, RNA interference mechanisms, apopotisisregulators and MAPK-signaling cascades. Infection of the C. elegansmutants can be characterized to specifically determine the relativecontributions of different innate immune system components to viraldisease and to identify how VSV infection is altered in these hosts. Forexample, if a mutant is infected with such as the defensin-likeantimicrobial peptides, one fo several outcomes can be envisioned. If agene is not involved in disease development or protection, then theresult should look identical to that seen wild-type or normal C.elegans. However, if the gene is involved in disease development thenthe disease symptoms should not appear or will be less severe in theinfected mutant (which is defective in this gene) than in the wild-typeor normal C. elegans. If the gene is involved in disease protection suchas immunity, then the disease symptoms should be more severe in theinfected mutant than in the wild-type or normal C. elegans. In thepresent invention, infection by rde-1 mutants led to more cells beinginfected and VSV gene expression as detected by the intensity of greenfluorescent protein expression in each cell was higher than in theinfected normal C. elegans (see FIGS. 6 a/b).

7. EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention.

7.1 C. elegans Infection Using Different Viruses

C. elegans were infected with VSV, vaccinia virus, catfish channel virus(CCV), and baculovirus. Vaccinia is a poxvirus. CCV is a herpes viruscapable of replication at 25° C. Baculoviruses are insect viruses thatare capable of replicating in other invertebrate hosts.

The infections gown in liquid media at 25° C. for 24 hours were examinedfor disease phenotypes including decreased numbers of progeny,alteration in larval development or adult life-span, muscleabnormalities leading to uncoordinated movement or egg-laying defectsand death.

7.2 C. elegans Infections Using Infected HeLa Cell Monolayers

Disease phenotypes were observed in C. elegans that feed on VSV-infectedHeLa cells where the infected monolayer grown in Dulbecco Modificationof Eagle Medium (DMEM) supplemented with 5% fetal calf serum provided asource of virons to infect C. elegans. Cells were infected with VSV at amultiplicity of 0.1 to 0.3 infectious virus particles per cell overnightat 37° C. Wild-type strain C. elegans larvae at the LA stage were addeddirectly to the media and maintained at 25° C. Negative controls wereuninfected HeLa cells.

After seven days, infected samples demonstrated a decrease in the numberof nematodes between 50% and 70% of the uninfected controls.

7.3 VSV Infections by VSV in Worm Growth Media

C. elegans cultured in worm growth (WG) media (10 mM NaCl, 10 mM KH₂PO₄;pH 6.0; 10 mM Potassium citrate; 1× trace metals; 3 mM CaCl₂; 3 mMMgSO₄; 13 μM cholesterol) were exposed to VSV in a cell-freesupernatant.

OP50, an auxotrophic strain of Escherichia coli deficient in uracilproduction was used as a food source for C. elegans. HEPES buffer (10mM, pH 7.0) was added to all samples to maintain a constant pH. VSVstocks were purified and resuspended in phosphate buffered saline (PBS)to remove all serum and cellular debris.

Stage L3 larvae were added to individual wells of 12-well plates. Eachwell contained 750 μl of media supplemented with one-tenth total volumeof an overnight OP50 culture in LB and 10⁶ PFU/ml VSV or an equal volumeof PBS. After three days at 25° C., the resulting populations werecounted and progeny were scored by developmental stage. All progeny werein the F1 generation.

The overall population size was significantly reduced in the presence ofVSV and the developmental stages of progeny were skewed toward the earlylarval stages (see FIG. 1). Since there were no infected HeLa cellspresent, disease was due to the presence of VSV. E. coli OP50 present inthe wells were alive and residual uracil in the LB media allowed forsome bacterial growth but the same results described in FIG. 1 wereobserved when heat-inactivated bacteria were used as a food source.

7.4 UV-inactivated VSV

A series of infections using UV-inactivated VSV were performed todetermine whether the observed disease phenotype required the presenceof infectious virus or was due to toxicity of viral proteins. One set ofnematodes was exposed to 10⁶ PFU/mL equivalent doses of UV-inactivatedVSV. Contrary to infectious VSV, the presence of UV-inactivated VSV hadno effect on the total population size after three days of exposure (seeFIG. 2). The developmental stage was skewed compared to uninfectedsamples but to a much lower degree than in samples containing infectiousvirus.

7.5 Assays for Viral Infection and Replication

7.5.1 Confocal Microscopy

A gene encoding green fluorescent protein (GFP) or its red derivative(dsRED) has been utilized as a marker for gene expression in prokaryoticand eukaryotic cells. GFP expressed in a heterologous prokaryotic oreukaryotic host produces a protein capable of fluorescence. C. eleganswas infected with a recombinant strain of VSV that carried GFP or dsREDin the viral genome. The presence of GFP indicated transcription of theviral RNA and expression of virus-encoded proteins because GFP is not avirus structural protein and is not packaged in the VSV virion.Morphologic changes of nematodes exposed to VSV included an increase influorescence of the intestinal lysosomes and separation of theintestinal lumen from the outer cuticle layers in nematodes exposed toVSV.

7.5.2 Antibody Staining

Antibodies generated against vesicular stomatitis virus G protein (VSV Gprotein); N and M proteins were utilized to detect for the production ofviral protein in infected C. elegans. The infected C. elegans cells werefixed with 4% paraformaldehyde incubated with antibody against VSVprotein and secondary antibody then examined by fluorescent microscopy.

7.5.3 RNA Analysis

RNA was isolated from virus infected C. elegans and baby hamster kidneycells (BHK) cell. Infected and uninfected BHK cells were used aspositive and negative assay controls, respectively. cDNA synthesis wasmade using oligonucleotide primers that specifically hybridized with Nprotein MnRNA transcripts at the 3′ terminus and not to the viral RNAgenome. The resultant cDNA sequences were amplified by Polymerase ChainReaction (PCR) using oligonucleotide primers specific for N protein genesequences. PCR signals we observed only in the infected C. elegans andBHK cells (see FIG. 5).

7.6 VSV infection of C. elegans Cells in Culture

Cells were isolated from C. elegans embryos and grown in culture inLeibovitz's L-15 media supplemented with 5% fetal calf serum at 25° C.Recombinant VSV expressing GFP was added to the media. The cells wereincubated with the recombinant virus for 12 hours at 25 ° C. at amultiplicity of 10 infectious unit per cell. At 48 hour post-infection,the cells were examined by fluorescent microscopy for GFP expression(see FIG. 3).

7.7 VSV Infection of Transgenic C. elegans Cells in Culture

Cells were isolated from transgenic C. elegans (strain PD4251) embryosas in Section 7.6. C. elegans strain PD4251 is a transgenic nematodeline that has GFP incorporated into the C. elegans genome and under theregulation of the myo-3 promoter. Thus, GFP expression is observed onlyin cells which activate the myo-3 promoter, including the body wallmuscle cells. Recombinant VSV expressing the dsRED form of GFP was addedto the media and the cells were examined 48-96 hours post-infection byfluorescent microscopy. The detection of GFP fluorescence was used toidentify muscle cells and dsREd fluorescence detection to identifyinfected cells.

Both green and red fluorescence was detected in the cell culture.However, the fluorescent signals did not co-localize (see FIG. 4). Ayellow signal would represent the presence of GFP from the muscle celland dsRED protein from VSV infection in the same cells. Thisdemonstrates that under these conditions the VSV is not infecting musclecells but infecting other cell types in the C. elegans.

7.8 VSV Infection of Mutant C. elegans Cells in Culture

Cells were isolated from mutant (strain WM27) and wild type C. elegansembryos as in Section 7.6. C. elegans strain WM27 carries the mutantrde-1 (ne219) allele which results in Rde-1 deficiency. Rde-1 isrequired in the RNA interference pathway and C. elegans strain WM27 isdefective in the RNA interference pathway. Recombinant VSV expressingGFP was added to the media and the cells were examined 48-96 hourspost-infection by fluorescent microscopy to detect GFP fluorescence.

GFP fluorescence was observed in a greater number of rde-1 mutant cellsthan wild type cells and the fluorescent intensities were higher in therde-1 mutant cells (see FIGS. 6 a/b). This suggests that the rde-1strain is more susceptible to VSV infection that the wild type C.elegans strain.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes. Many modifications and variations of thisinvention can be made without departing from its spirit and scope, aswill be apparent to those skilled in the art. The specific embodimentsdescribed herein are offered by way of example only, and the inventionis to be limited only by the terms of the appended claims, along withthe full scope of equivalents to which such claims are entitled.

REFERENCES

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1. A method for detecting disease conditions in C. elegans comprising:(i) infecting C. elegans with an animal virus wherein the animal virusis selected from the group consisting of wild-type, mutant, recombinantand pseudo-type; and (ii) detecting a change in phenotype of theinfected C. elegans wherein said change in phenotype not detected inuninfected C. elegans is indicative of disease conditions in theinfected C. elegans.
 2. A method for detecting disease conditions in C.elegans comprising: (i) infecting C. elegans with a vesicular stomatitisvirus wherein vesicular stomatitis virus is selected from the groupconsisting of wild-type, mutant, recombinant and pseudo-type; and (ii)detecting a change in phenotype of the infected C. elegans wherein saidchange in phenotype not detected in uninfected C. elegans is indicativeof disease conditions in the infected C. elegans.
 3. A method accordingto claim 1 or 2, wherein the change in phenotype is selected from thegroup consisting of decreased numbers of progeny, alteration in larvaldevelopment, alteration in adult life span, muscle abnormalities leadingto uncoordinated movements, egg-laying defects and death.
 4. A methodaccording to claim 1 or 2, wherein C. elegans is selected from the groupconsisting of wild-type, mutant, recombinant and transgenic C. elegans.5. A method for identifying genes involved in the development of diseaseconditions in C. elegans comprising: (i) infecting C. elegans with ananimal virus wherein the animal virus is selected from the groupconsisting of wild-type, mutant, recombinant and pseudo-type; (ii)detecting a change in phenotype of the infected C. elegans wherein saidchange in phenotype not detected in uninfected C. elegans is indicativeof disease conditions in the infected C. elegans; and (iii) Identifyinggenes that are either induced or repressed by infection of the C.elegans.
 6. A method for identifying genes involved in the developmentof disease conditions in i C. elegans comprising: (i) infecting C.elegans with a vesicular stomatitis virus wherein vesicular stomatitisvirus is selected from the group consisting of wild-type, mutant,recombinant and pseudo-type; (ii) detecting a change in phenotype of theinfected C. elegans wherein said change in phenotype not detected inuninfected C. elegans is indicative of disease conditions in theinfected C. elegans; and (iii) identifying genes that are either inducedor repressed by infection of the C. elegans.
 7. A method according toclaim 5 or 6, wherein the change in phenotype is selected from the groupconsisting of decreased numbers of progeny, alteration in larval,development alteration in adult life span, muscle abnormalities leadingto uncoordinated movements, egg-laying defects and death.
 8. A methodaccording to claim 6 or 7, wherein C. elegans is selected from the groupconsisting of wild-type, mutant, recombinant and transgenic C. elegans.9. A method for screening agents that effect viral diseases in C.elegans comprising: (i) infecting C. elegans with an animal viruswherein the animal virus is selected from the group consisting ofwild-type, mutant, recombinant and pseudo-type; (ii) combining an agentwith the infected C. elegans; and (iii) determining the effect of theagent on a change in phenotype not detected in uninfected C. elegans.10. A method for screening agents that effect vesicular stomatitis virusdisease comprising: (i) infecting C. elegans with a vesicular stomatitisvirus wherein vesicular stomatitis virus is selected from the groupconsisting of wild-type, mutant, recombinant and pseudo-type; (ii)combining an agent with the infected C. elegans; and (iii) determiningthe effect of the agent on a change in phenotype not detected inuninfected C. elegans.
 11. A method according to claim 9 or 10, whereinthe change in phenotype is selected from the group consisting ofdecreased numbers of progeny, alteration in larval development,alteration in adult life span, muscle abnormalities leading touncoordinated movements, egg-laying defects and death.
 12. A methodaccording to claim 9 or 10, wherein C. elegans is selected from thegroup consisting of wild-type, mutant, recombinant and transgenic C.elegans.
 13. A method for testing an agent for effectiveness against aviral disease condition in C. elegans comprising: (i) infecting C.elegans with an animal virus wherein the animal virus is selected fromthe group consisting of wild-type, mutant, recombinant and pseudo-type,and wherein the C. elegans exhibits a disease condition phenotype; (ii)delivering the agent to the infected C. elegans; and (iii) analyzing theeffectiveness of the agent on the infected C. elegans wherein an agentthat diminishes the disease condition phenotype of the infected C.elegans is an agent that has effectiveness against the disease conditionin the infected C. elegans.
 14. A method for testing an agent foreffectiveness against a vesicular stomatitis virus disease condition inC. elegans comprising: (i) infecting C. elegans with a vesicularstomatitis virus wherein vesicular stomatitis virus is selected from thegroup consisting of wild-type, mutant, recombinant and pseudo-type, andwherein the C. elegans exhibits a disease condition phenotype; (ii)delivering the agent to the infected C. elegans; and (iii) analyzing theeffectiveness of the agent on the infected C. elegans wherein an agentthat diminishes the disease condition phenotype of the infected C.elegans is an agent that has effectiveness against the disease conditionin the infected C. elegans.
 15. A method according to claim 13 or 14,wherein the change in phenotype is selected from the group consisting ofdecreased numbers of progeny, alteration in larval development,alteration in adult life span, muscle abnormalities leading touncoordinated movements, egg-laying defects and death.
 16. A methodaccording to claim 13 or 14, wherein C. elegans is selected from thegroup consisting of wild-type, mutant, recombinant and transgenic C.elegans.